1. Field of the Invention
This invention relates to a method of predisposing mammals, especially humans, to accelerated tissue repair. More particularly, this invention is directed to a method of treating a mammal with transforming growth factor-beta before tissue injury to accelerate repair of the tissue.
2. Description of Related Disclosures
The beta transforming growth factors (TGF-.beta.s) are multifunctional cytokines, produced by many types of cells, including hematopoietic, neural, heart, fibroblast, and tumor cells, that can regulate the growth and differentiation of cells from a variety of tissue origins (Sporn et al., Science, 233: 532 (1986)) and stimulate the formation and elaboration of various stromal elements.
There are at least five forms of TGF-.beta. currently identified, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5. Suitable methods are known for purifying this family of TGF-.beta.s from various species such as human, mouse, green monkey, pig, bovine, chick, and frog, and from various body sources such as bone, platelets, or placenta, for producing it in recombinant cell culture, and for determining its activity. See, for example, R. Derynck et al., Nature, 316:701-705 (1985); European Pat. Pub. Nos. 200,341 published Dec. 10, 1986, 169,016 published Jan. 22, 1986, 268,561 published May 25, 1988, and 267,463 published May 18, 1988; U.S. Pat. No. 4,774,322; Cheifetz et al, Cell, 48: 409-415 (1987); Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); Dijke et al., Proc. Natl. Acad. Sci. (U.S.A.), 85: 4715-4719 (1988); Derynck et al., J. Biol. Chem., 261: 4377-4379 (1986); Sharples et al., DNA, 6: 239-244 (1987); Derynck et al., Nucl. Acids. Res., 15: 3188-3189 (1987); Derynck et al., Nucl. Acids. Res., 15: 3187 (1987); Derynck et al., EMBO J., 7: 3737-3743 (1988)); Seyedin et al., J. Biol. Chem., 261: 5693-5695 (1986): Madisen et al., DNA, 7: 1-B (198B): and Hanks et al., Proc. Natl. Acad. Sci. (U.S.A.), 85: 79-82 (1988), the entire contents of these publications being expressly incorporated by reference.
The activated form of TGF-.beta.1is a homodimer formed by dimerization of the carboxy-terminal 112 amino acids of a 390 amino acid precursor (Derynck et al., Nature, supra). Recombinant TGF-.beta.1 has been cloned (Derynck et al., Nature, supra) and expressed in Chinese hamster ovary cells (Gentry et al., Mol. Cell. Biol., 7: 3418-3427 (1987)).
TGF-.beta.2 has a precursor form of 414 amino acids and is also processed to a homodimer from the carboxy-terminal 112 amino acids that shares approximately 70% homology with the active form of TGF-.beta.1 (Marquardt et al., J. Biol. Chem., 262: 12127 (1987)). TGF-.beta.2 has been purified from porcine platelets (Seyedin et al., J. Biol. Chem., 262: 1946-1949 (1987)) and human glioblastoma cells (Wrann et al., EMBO J., 6: 1633 (1987)), and recombinant human TGF-.beta.2 has been cloned (deMartin et al., EMBO J., 6: 3673 (1987)).
TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5, which are the most recently discovered forms of TGF-.beta., were identified by screening cDNA libraries. The putative protein products of these three genes have not been isolated from natural sources, although Northern blots demonstrate expression of the corresponding mRNAs. Human and porcine TGF-.beta.3 have been cloned and described previously (Derynck et al., EMBO J.,7: 3737-3743 (1988), ten Dijke et al., Proc. Natl. Acad. Sci. USA, 85: 4715 (1988)). TGF-.beta.4 and TGF-.beta.5 were cloned from a chicken chondrocyte cDNA library (Jakowlew et al., Molec. Endocrinol., 2: 1186-1195 (1988)) and from a frog oocyte cDNA library, respectively The frog oocyte cDNA library can be screened using a probe derived from one or more sequences of another type of TGF-.beta.. TGF-.beta.4 mRNA is detectable in chick embryo chondrocytes, but is far less abundant than TGF-.beta.3 mRNA in developing embryos or in chick embryo fibroblasts. TGF-.beta.5 mRNA is expressed in frog embryos beyond the neurula state and in Xenopus tadpole (XTC) cells.
TGF-.beta. has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. TGF-.beta. is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (Sporn et al., supra). For a general review of TGF-.beta. and its actions, see Sporn et al., J. Cell Biol., 105: 1039-1045 (1987), Sporn and Roberts, Nature, 332: 217-219 (1988), and Roberts et al., Recent Progress in Hormone Research, 44: 157-197 (1988).
Natural TGF-.beta.1 is made predominantly, if not exclusively, in a biologically latent form, which can be activated in vitro by denaturants such as urea, heat, plasmin, high salt, endoglycosidase F, capthepsin D, type IV collagenase, cocultured endothelial cells and pericytes, plasminogen activators such as urokinase, stimulated osteoclasts, or extremes of pH. See, e.g., Pircher et al., Canc. Res., 44: 5538-5543 (1984) re latent TGF-.beta. from nontransformed and Kirsten sarcoma virus-transformed normal rat kidney cells; Antonelli-Orlidge et al., Proc. Natl. Acad. Sci. USA, 86: 4544-4548 (1989) re latent TGF-.beta. from pericytes and capillary endothelial cells; Lawrence et al., Biochem. Biophys. Res. Commun., 133: 1026-1034 (1985) re latent TGF-.beta. from chicken embryo fibroblasts; Oreffo et al., Biochem. Biophys. Res. Commun., 158: 817-823 (1989) re latent TGF-.beta. from murine bone organ cultures; Keski-Oja et al., J. Cell Biol., 107: (6 Part 3), 1988, 50a re latent TGF-.beta. from human lung adenocarcinoma cell line; Miyazono and Heldin, J. Cell. Biochem. SuDD. 0 (13 part B) 1989, p. 92 and Miyazono and Heldin, Nature, 338: 158-160 (1989) re latent TGF-.beta. from human platelets and its carbohydrate structure; and Pircher et al., Biochem. Biophys. Res. Commun., 136: 30-37 (1986) re latent TGF-.beta. from human blood platelets. See also Lawrence et al., J. Cell. Physiol., 121: 184-188 (1984); Kryceve-Martinerie et al., Int. J. Cancer, 35: 553-558 (1985); Brown et al., "TGF-.beta.", N.Y. Acad. Sci. Meeting Abstract, May 18-20, 1989; Danielpour et al., J. Cell. Physiol., 138: 79-86 (1989); Wakefield et al., J. Biol. Chem., 263: 7646-7654 (1988); and Miyazono et al., J. Biol. Chem., 263: 6407-6415 (1988 ).
Several groups have characterized the latent form of TGF-.beta.1secreted by human platelets. Pircher et al., supra, stated that it has an apparent molecular weight of 400 Kd. More recently, it has been characterized as a three-component complex of about 235 Kd, wherein the active TGF-.beta.l (25 Kd dimer) is non-covalently associated with the remainder of the processed precursor (75 Kd dimer), which in turn is disulfide-bonded to an unrelated protein of 125-160 Kd (Wakefield et al., J. Biol. Chem., 263, supra; Miyazono et al., supra; Miyazono et al., J. Cell Biochem. Supp., 0 (12 part A), 1988, p. 200; Wakefield et al., J. Cell. Biochem. Suppl., 11A: 0, 46 (1987)).
The function of the binding protein of 125-160 Kd remains to be elucidated. Recent characterizations indicate that it contains at least 14 EGF-like repeats and six potential N-glycosylation sites and calcium binding domains (Kanzaki et al., "TGF-.beta.", N.Y. Acad. Sci. meeting abstract, May 18-20, 1989; Miyazono, "TGF-.beta.", N.Y. Acad. Sci. meeting abstract, May 18-20, 1989). Latent TGF-.beta. secreted by many cells in culture has a similar structure (Wakefield et al., J. Biol. Chem., supra), and this is the form in which TGF-.beta.1 is probably perceived initially by target cells in vivo. It has been suggested that the precursor remainder of TGF-.beta. may have an important independent biological function based on conservation of sequences in the precursor region (Roberts et al., Recent Progress in Hormone Research, supra). Additionally, a mutation at position 33 of precursor TGF-.beta.1 is reported to increase the yield of mature TGF-.beta.l, and dimerization of the precursor "pro" region is suggested as necessary to confer latency (Brunner et al., J. Biol. Chem., 264: 13660-13664 (1989)).
Normal repair of tissue is a complex, sequential process involving many cell types. Fibroblasts, inflammatory cells, and keratinocytes all function in an integrated manner to promote cell division, differentiation, and migration. These processes in turn lead to enhanced connective tissue deposition and angiogenesis. Recent data suggest that these processes may be mediated both in an autocrine and paracrine manner by peptide growth factors such as TGF-.beta. (Postlethwaite et al., J. Exp. Med., 165: 251-256 (1987); Assoian et al., Nature, 308: 804-806 (1984)). Levels of endogenous TGF-.beta. have been reported to increase transiently in wound chambers of the rat (Cromack et al., J. Surg. Res., 42: 622-628 (1987)). Also, a crude extract of platelets containing multiple growth factors promoted healing of chronic skin ulcers (Knighton et al., Ann Surg., 204: 322-330 (1986)). The results of these studies indirectly support the hypothesis that normal healing is mediated by locally produced peptide growth factors.
In vivo, TGF-.beta.1 causes granulation tissue to form when injected intradermally (Roberts et al., Proc. Nat. Acad. Sci. USA, 83: 4167-4171 (1986); Sporn et al., Science, 219: 1329-1331 (1983)). In vitro, TGF-.beta.1 stimulates the expression of fibronectin and collagen type I, in part mediated via increased levels of mRNA, and increases the deposition of fibronectin into the pericellular matrix (Wrana et al., Eur. J. Biochem., 159: 69-76 (1986); Ignotz and Massague, J. Biol. Chem., 261: 4337-4345 (1986); Fine and Goldstein, J. Biol. Chem., 262: 3897-3902 (1987); Ignotz et al., J. Biol. Chem., 262: 6443-6446 (1987); Raghow et al., J. Clin. Invest., 79: 1285-1288 (1987); Varga and Jimeniz, Biochem. Biophys. Res. Commun. 138: 974-980 (1986)).
A single application of TGF-.beta. in collagen vehicle to incisions in normal rats significantly increased tensile strength compared with untreated or collagen vehicle treated incisions (Mustoe et al., Science, 237: 1333-1336 (1987)). See also Brown et al., Ann. Surg., 208: 788-794 (1988). In another study it was reported that TGF-.beta. treatment reversed doxorubicin depressed uptake of hydroxyproline and thymidine in wound chambers in rats, suggesting that TGF-.beta. might enhance the strength of the incisions by stimulating proliferation of cells and enhancing collagen synthesis (Grotendorst et al., J. Clin. Invest., 76: 2323-2329 (1985)).
These results were extended using an animal model that more closely approximates healing of surgical incisions (Curtsinger et al., Surgery, Gynecology & Obstetrics, 168: 517-522 (1989)). It was hypothesized that because TGF-.beta. is a potent chemoattractant for human fibroblasts (Postlethwaite et al., supra,) and stimulates collagen synthesis in cultures of renal fibroblasts in rats (Roberts et al., Proc. Natl. Acad. Sci. USA, supra), it may increase tensile strength by directly stimulating production of collagen by fibroblasts or by attracting inflammatory cells that may release peptide growth factors into the wounded area (Madtes et al., Cell, 53: 285-293 (1988); Morhenn, Immunol. Today, 9: 104-107 (1988)).
In addition to the scientific literature, the patent literature has also disclosed that TGF-.beta. is useful in treating existing traumata when administered systemically or applied topically to the traumatized tissue, with promotion of rapid proliferation of cells, particularly fibroblast cells (see, e.g., EP 128,849; EP 105,014; U.S. Pat. Nos. 4,843,063; 4,774,322; 4,774,228; and 4,810,691). There is, however, also a need for an agent that predisposes mammals to accelerated tissue repair before the mammals have been subjected to trauma.
Accordingly, it is an object of the present invention to provide a method for treating mammals that have not yet experienced tissue damage to promote accelerated proliferation of the cells surrounding the traumata and consequently rapid healing.
This object and other objects will become apparent to one of ordinary skill in the art.